• 동의생리병리학회지
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• 제19권3호
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• pp.640-646
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• 2005

This study was performed for the investigation of anticancer effects of Siegesbeckia glabrescens(SG) on HepG2 cells, a human hepatoma cell line. In the previous study, we examined the involvement of nitric oxide (NO) on anti-proliferative and apoptotic efficacy of SG in vascular smooth muscle cells. The possible mechanism of the apoptotic effects of SG was investigated in HepG2 cells. SG showed potent cytotoxic activity in HepG2 but not chang cells, liver normal cells. SG treatment caused morphological change such as cell shrinkage, nuclei condensation and cell blebbing in HepG2 cells. SG also increased the nitrite production of HepG2 cells in a dose-dependent manner. Furthermore, L-NNA treatment inhibited the anti-proliferative effect of SG. From RT-PCR, SG decreased Bcl-2 but no affected on Bax. Western blot for procaspase-3 and COX-2 showed that degradation of procaspase-3 protein level or inhibition of COX-2 protein expression by SG treatment. In addition, the apoptotic effect of SG was also demonstrated by DNA laddering. In conclusion, SG-induced HepG2 cells death can occur via apoptosis which was dose-dependent, and associated with apoptosis-related Bcl-2/Bax gene expressions, COX-2 inhibition, caspase-3 activation and NO pathway. These results suggest that SG is potentially useful as a chemotherapeutic/chemopreventive agent in hepatocellular carcinoma.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1998년도 Proceedings of UNESCO-internetwork Cooperative Regional Seminar and Workshop on Bioassay Guided Isolation of Bioactive Substances from Natural Products and Microbial Products
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• pp.198-198
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• 1998

Muc1 mucin is found in a variety of epithelial tissue and is overexpressed in several epithelial cancer. Recently it is alsol reported that primary Hamster tracheal surface epithelial(HTSE) cells express Muc1 protein and cDNA encoding HTSE muc1 protein has been cloned. Although numerous monoclonal antibodies (mAbs) to human muncins, particularly Muc1 have been produced, no such antibodies to murine Muc1 have been described. We now describe monoclonal antibody, called mAb M1CT, produced to C-terminal region of HTSE Muc1 protein by immunising mice with a glutathion-s-transferase linked fusion protein. In this study, using this antibody(mAb M1CT) we investigated the effect of RA on the expression of Muc1 in HTSE cells. Retinoic acid(RA) plays an essential role in maintaining normal differentiation of tracheal epithelial cells. With RA-deficiency tracheocytes undergo squamous metaplasia, an abnormal differentiation that can be reversed by RA. We had primary culture of HTSE cells under different concentrations of RA. Culture was maintained until the direction of differentiation was determined. Then Western blot analysis with mAb M1CT was performed with the cell lysates from the culture. The expression of Muc1 protein was decreased in dose-dependent manner as the concentration of retinoic acid was decreased. Our result indicates that the expression of Muc1 protein is coordinately regulated with airway mucous cell differentiation by RA pathway. And the antibody, mAb M1CT, produced in this study should provide useful tool to study the expression of Muc1 mucin in differentiation process or disease.

• Toxicological Research
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• 제31권2호
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• pp.97-104
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• 2015

The skin exposure to solar irradiation and photoreactive xenobiotics may produce abnormal skin reaction, phototoxicity. Phototoxicity is an acute light-induced response, which occurs when photoreacive chemicals are activated by solar lights and transformed into products cytotoxic against the skin cells. Multifarious symptoms of phototoxicity are identified, skin irritation, erythema, pruritis, and edema that are similar to those of the exaggerated sunburn. Diverse organic chemicals, especially drugs, are known to induce phototoxicity, which is probably from the common possession of UV-absorbing benzene or heterocyclic rings in their molecular structures. Both UVB (290~320 nm) and UVA (320~400 nm) are responsible for the manifestation of phototoxicity. Absorption of photons and absorbed energy (hv) by photoactive chemicals results in molecular changes or generates reactive oxygen species and depending on the way how endogenous molecules are affected by phototoxicants, mechanisms of phototoxcity is categorized into two modes of action: Direct when unstable species from excited state directly react with the endogenous molecules, and indirect when endogeneous molecules react with secondary photoproducts. In order to identify phototoxic potential of a chemical, various test methods have been introduced. Focus is given to animal alternative test methods, i.e., in vitro, and in chemico assays as well as in vivo. 3T3 neutral red uptake assay, erythrocyte photohemolysis test, and phototoxicity test using human 3-dimensional (3D) epidermis model are examples of in vitro assays. In chemico methods evaluate the generation of reactive oxygen species or DNA strand break activity employing plasmid for chemicals, or drugs with phototoxic potential.

• Molecular & Cellular Toxicology
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• 제3권2호
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• pp.137-144
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• 2007

The mechanism of cytotoxicity of doxifluridine, a prodrug fluorouracil (5-FU), has been ascribed to the misincorporation of fluoropyrimidine into RNA and DNA and to the inhibition of the nucleotide synthetic enzyme thymidylate synthase. Increased understanding of the mechanism of 5-FU has led to the development of strategies that increases its anticancer activity or predicts its sensitivity to patients. Using GeneChip?? Rhesus Macaque Genome arrays, we analyzed gene expression profiles of doxifluridine after two weeks repeated administration in cynomolgus monkey. Kegg pathway analysis suggested that cytoskeletal rearrangement and cell adhesion remodeling were commonly occurred in colon, jejunum, and liver. However, expression of genes encoding extracellular matrix was distinguished colon from others. In colon, COL6A2, COL18A1, ELN, and LAMA5 were over-expressed. In contrast, genes included in same category were down-regulated in jejunum and liver. Interestingly, MMP7 and TIMP1, the key enzymes responsible for ECM regulation, were overexpressed in colon. Several studies were reported that both gene reduced cell sensitivity to chemotherapy-induced apoptosis. Therefore, we suggest they have potential as target for modulation of 5-FU action. In addition, the expression of genes which have been previously known to involve in 5-FU pathway, were examined in three organs. Particularly, there were more remarkable changes in colon than in others. In colon, ECGF1, DYPD, TYMS, DHFR, FPGS, DUT, BCL2, BAX, and BAK1 except CAD were expressed in the direction that was good response to doxifluridine. These results may provide that colon is a prominent target of doxifluridine and transcriptional profiling is useful to find new targets affecting the response to the drug.

• 미생물학회지
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• 제35권1호
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• pp.13-17
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• 1999

Shigella sonnei 는 인체의 장내 감염을 일으키는 병원균의 일종이며, 본 연구에 사용된 S.sonnei KNIH104S는 국내에서 쉬겔라증을 나타내는 환자로부터 분리하였다. S. sonnei KNIH104s의 염색체로부터 aspartate $\beta$-semial-dehyde dehydrogenase를 암호화하는 asd 유전자를 포함하는 1.7 kb BamHI 절편을 pBluescript SK(+) 벡터를 이용하여 클로닝하였으며 pSAB17이라 명명하였다. asd 결실돌연변이주인 E.coli $\chi$6097은 세포벽을 구성하는 중요한 성분인 DL-$\alpha$, $\varepsilon$-diaminopimelic acid가 없는 Luria-Bertani 배지에서 생장함을 확인하였다. 클로닝된 asd 유전자의 염기서열 분석결과 ATG 개시코돈 및 TAA 종결코돈을 지니는 1,104 bp 로 이루어져 있으며, 여기서 유추한 아미노산은 367개로 분자량 40.0 kDa 의 폴리펩타이드를 만들어내고 있다. 염기서열은 대장균의 asd 유전자와 한 부위에서 다르게 나타났지만 아미노산의 서열은 동일함을 알 수 있었다. 그리고 pBluescript SK(+) 벡터와 본 연구에서 클로닝된 asd 유전자를 이용하여 balanced-lethal vector인 pSKA47 및 pSKA47A를 제조하였다.

• 미생물학회지
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• 제36권1호
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• pp.46-51
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• 2000

장티푸스는 Salmonella typhi에 의해 유발되는 장감염성 질환으로 사람과 동물에 공통되는 질병이다. 본 연구에서는 국립보건원과 공동연구를 수행하여 한국형 장티푸스 유발균인 S. typhi KNIH100을 분리하였다. 분리된 S. typhi KNIH100의 염색체 DNA로부터 방향족 아미노산의 생합성에 관여하는 효소인 5-enolpyruvylshikimate-3-phosphate synthetase를 암호화하는 aroA 유전자를 포함하는 약 5.0 kb의 SalI절편을 pBluescriptII SK(+) vector와 aroA 돌연변이주인 E. coli CGSC2829를 이용하여 클로닝하였다. 그리고 이 클론을 pSAL80이라 명명하였다. 클로닝된 재조합 plasmid인 pSAL80에는 ATG 개시코돈과 TGA 종결코돈을 포함하는 1,284 염기로 구성된 aroA 유전자가 위치하고 있었으며, 다른 장내세균과 마찬가지로 serC와 하나의 오페론을 구성하고 있음을 밝혔다. 또한 S. typhi Ty2, S. typhimurium, 그리고 E. coli 등 다른 장내세균의 aroA 유전자와 상동성을 비교하여 본 결과 각각 99%, 95%, 77%의 상동성을 나타내었다.

• 디지털융복합연구
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• 제14권4호
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• pp.349-353
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• 2016

융복합 패러다임의 도입은 방대한 유전체 정보의 분석을 위한 컴퓨팅 기술의 연구 및 개발 또한 활발히 진행되고 있다. 최근 유전체 분석 서비스 유형은 개인의 유전체 정보(personal genome analysis)를 읽어서 특정 질환들의 발병 확률 등을 알려주고, 해당 질병을 예방할 수 있도록 식습관, 라이프 스타일등의 변화를 꾀하도록 맞춤형의 서비스를 제공하고 있다. 생물의 특성을 결정하는 정보는 유전자이며, 이 유전자는 DNA 염기서열에 따라 결정되므로, 유전체 정보의 분석기술은 정확하고 빠르게 수행되어야 한다. 정확한 유전체 분석을 빠르게 수행하기위해 K-Mean 클러스터링 기법을 활용하였으며, 코돈 데이타 패턴을 추출하여 유전체 정보 분석에 적용하였다. 또한, 미토콘드리아 데이타군을 실험사례로 제공한다. 본 연구의 결과, 제공된 분석 데이타를 통해 기존의 문자열 형태의 유전체 분석 기법을 이미지 패턴 형태로 추출이 가능하며, 패턴형태의 이미지는 분석시간의 단축과 정확도를 높인다.

• KSBB Journal
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• 제24권3호
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• pp.279-286
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• 2009

방선균은 다양한 생리활성 물질을 이차대사산물로 생산하는 산업적으로 매우 유용한 미생물이다. 이에 따라 많은 연구진들이 방선균에 대한 분자생물학적 연구와 산업적 이용에 대한 연구들을 수행하고 있다. 방선균 중에서도 S. avermitilis는 강력한 구충효과가 있는 avermectin을 생산하지만, 또한 포유동물 세포의 미토콘드리아에서 산화적 인산화반응을 억제하는 oligomycin도 함께 생성된다. 따라서 S. avermitilis에서 oligomycin의 생성을 제거시키기 위하여 oligomycin synthetase gene을 disruption 시키기 위한 연구를 수행하였다. 이를 위하여 S. avermitilis로부터 cloning 한 oligomycin synthetase gene (olmA5)의 중앙부분에 apramycin resistance gene을 끼워 넣어 integration vector로 구축한 후에 S. avermitilis의 chromosomal DNA와의 homologous recombination에 의하여 olmA5 gene의 disruption을 유도하였다. Disruption mutants (olmA5::apra)는 PCR을 통해 olmA5 gene의 위치에 apramycin resistance gene이 존재하는 것으로 확인하였고, 또한 HPLC 분석을 통해 oligomycin 생합성이 완전히 제거된 것임을 확인하였다. 그러나 disruption mutant (olmA5::apra)를 이용하여 avermectin 만을 생산할 수 있었으나, avermectin의 생산량에는 거의 변화가 없었다. 이러한 mutants는 산업적으로 avermectin을 생산하기 위한 균주 개량의 훌륭한 source가 될 수 있을 것이다.

• Genomics & Informatics
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• 제10권3호
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• pp.194-199
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• 2012

In addition to single-nucleotide polymorphisms (SNP), copy number variation (CNV) is a major component of human genetic diversity. Among many whole-genome analysis platforms, SNP arrays have been commonly used for genomewide CNV discovery. Recently, a number of CNV defining algorithms from SNP genotyping data have been developed; however, due to the fundamental limitation of SNP genotyping data for the measurement of signal intensity, there are still concerns regarding the possibility of false discovery or low sensitivity for detecting CNVs. In this study, we aimed to verify the effect of combining multiple CNV calling algorithms and set up the most reliable pipeline for CNV calling with Affymetrix Genomewide SNP 5.0 data. For this purpose, we selected the 3 most commonly used algorithms for CNV segmentation from SNP genotyping data, PennCNV, QuantiSNP; and BirdSuite. After defining the CNV loci using the 3 different algorithms, we assessed how many of them overlapped with each other, and we also validated the CNVs by genomic quantitative PCR. Through this analysis, we proposed that for reliable CNV-based genomewide association study using SNP array data, CNV calls must be performed with at least 3 different algorithms and that the CNVs consistently called from more than 2 algorithms must be used for association analysis, because they are more reliable than the CNVs called from a single algorithm. Our result will be helpful to set up the CNV analysis protocols for Affymetrix Genomewide SNP 5.0 genotyping data.

• Genomics & Informatics
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• 제10권3호
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• pp.145-152
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• 2012

Chromatin structure and dynamics that are influenced by epigenetic marks, such as histone modification and DNA methylation, play a crucial role in modulating gene transcription. To understand the relationship between histone modifications and regulatory elements in breast cancer cells, we compared our chromatin immunoprecipitation sequencing (ChIP-Seq) histone modification patterns for histone H3K4me1, H3K4me3, H3K9/16ac, and H3K27me3 in MCF-7 cells with publicly available formaldehyde-assisted isolation of regulatory elements (FAIRE)-chip signals in human chromosomes 8, 11, and 12, identified by a method called FAIRE. Active regulatory elements defined by FAIRE were highly associated with active histone modifications, like H3K4me3 and H3K9/16ac, especially near transcription start sites. The H3K9/16ac-enriched genes that overlapped with FAIRE signals (FAIRE-H3K9/14ac) were moderately correlated with gene expression levels. We also identified functional sequence motifs at H3K4me1-enriched FAIRE sites upstream of putative promoters, suggesting that regulatory elements could be associated with H3K4me1 to be regarded as distal regulatory elements. Our results might provide an insight into epigenetic regulatory mechanisms explaining the association of histone modifications with open chromatin structure in breast cancer cells.